HAVCR1 Knockout Cell Line (HEK 293)

T-cell immunoglobulin mucin family member-1 (TIM-1, also known as HAVCR1/KIM-1) is a transmembrane glycoprotein that has been reported to act as an entry receptor for multiple flaviviruses including Zika virus (ZIKV). The post-translational regulation of TIM-1 and its effects on ZIKV infection are unclear. In this study, we identified the membrane-associated RING-CH-type finger (MARCH) E3 ubiquitin ligase family members MARCH2 and MARCH3 as critical negative regulators of TIM-1 under physiological conditions. MARCH2 and MARCH3 associate with TIM-1 and mediate its K48-linked polyubiquitination at K338 and K346 respectively, leading to subsequent proteasomal degradation. While deficiency of either MARCH2 or MARCH3 modestly increases TIM-1 levels and enhances ZIKV infectivity, double knockout of MARCH2/3 has a more dramatic effect. Double knockout of MARCH2/3 increased ZIKV infectivity in wild-type but not TIM-1 knockout cells, and reconstitution of TIM-1 into TIM-1-deficient cells increases ZIKV infectivity to a higher degree than reconstitution with wild-type TIM-1. Knockout of either MARCH2 or MARCH3 increased ZIKV infectivity and pathogenesis in mice, whereas double knockout of MARCH2/3 has a more dramatic effect. These findings suggest that MARCH2 and MARCH3 target TIM-1 for K48-linked polyubiquitination and proteasomal degradation, thereby acting as redundant host restriction factors to limit ZIKV infection and pathogenesis.

Orthoflaviviruses cause a major threat to global public health, and no antiviral treatment is available yet. Zika virus (ZIKV) entry, together with many other viruses, is known to be enhanced by phosphatidylserine (PS) receptors such as T-cell immunoglobulin mucin domain protein 1 (TIM-1). In this study, we demonstrate for the first time, using cell-based electrical impedance (CEI) biosensing, that ZIKV entry is also enhanced by expression of CD300a, another PS receptor. Furthermore, inhibiting CD300a in immature monocyte-derived dendritic cells partially but significantly inhibits ZIKV replication. As we have previously demonstrated that CEI is a useful tool to study Orthoflavivirus infection in real time, we now use this technology to determine how these PS receptors influence the kinetics of in vitro ZIKV infection. Results show that ZIKV entry is highly sensitive to minor changes in TIM-1 expression, both after overexpression of TIM-1 in infection-resistant HEK293T cells, as well as after partial knockout of TIM-1 in susceptible A549 cells. These results are confirmed by quantification of viral copy number and viral infectivity, demonstrating that CEI is highly suited to study and compare virus-host interactions. Overall, the results presented here demonstrate the potential of targeting this universal viral entry pathway.

T-cell immunoglobin and mucin domain protein-1 (TIM-1) mediates entry of chikungunya virus (CHIKV) into some mammalian cells through the interaction with envelope phospholipids. While this interaction enhances entry, TIM-1 has been shown to tether newly formed HIV and Ebola virus particles, limiting their efficient release. In this study, we investigate the ability of surface receptors such as TIM-1 to sequester newly budded virions on the surface of infected cells. We established a luminescence reporter system to produce chikungunya viral particles that integrate nano-luciferase and easily quantify viral particles. We found that TIM-1 on the surface of host cells significantly reduced CHIKV release efficiency in comparison to other entry factors. Removal of cell surface TIM-1 through direct cellular knock-out or altering the cellular lipid distribution enhanced CHIKV release. Over the course of infection, CHIKV was able to counteract the tethering effect by gradually decreasing the surface levels of TIM-1 in a process mediated by the nonstructural protein 2. This study highlights the importance of phosphatidylserine receptors in mediating not only the entry of CHIKV but also its release and could aid in developing cell lines capable of enhanced vaccine production. IMPORTANCE:Chikungunya virus (CHIKV) is an enveloped alphavirus transmitted by the bites of infectious mosquitoes. Infection with CHIKV results in the development of fever, joint pain, and arthralgia that can become chronic and last for months after infection. Prevention of this disease is still highly focused on vector control strategies. In December 2023, a new live attenuated vaccine against CHIKV was approved by the FDA. We aimed to study the cellular factors involved in CHIKV release, to better understand CHIKV's ability to efficiently infect and spread among a wide variety of cell lines. We found that TIM-1 receptors can significantly abrogate CHIKV's ability to efficiently exit infected cells. This information can be beneficial for maximizing viral particle production in laboratory settings and during vaccine manufacturing.